Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia

ABSTRACT Clostridium species have gained attention in industrial and medical applications, and the development of genetic tools has enabled the advancement of the CRISPR-Cas systems for these bacteria. Compared to the primarily used Cas9 from Streptococcus pyogenes, the utilization of Cas12a (previously known as Cpf1) proteins remains incomplete in clostridia, although they exhibit potential advantages, including T-rich recognition for Clostridium genomes and lower toxicity. In this study, we expanded the CRISPR-Cas tools in clostridia by establishing a CRISPR-Cas12a system with two different cas12a genes (Ascas12a from Acidaminococcus and Fncas12a from Francisella novicida). The optimized tetracycline-inducible systems were initially determined by the glucuronidase reporter and were used to drive expression of the cas12a genes and crRNAs. Our results demonstrate that the CRISPR-FnCas12a system offers flexible target selection in clostridia, and a specific folding pattern of the precursor crRNA is important to enable high mutation generation efficiency. By using sacB (encoding levansucrase) as a negative marker for plasmid curing and determining the optimal size of the donor DNA template for gene integration in the CRISPR-FnCas12a system, we achieved highly efficient and rapid genome modification, exemplified by the successful engineering of clostridia (Clostridium butyricum and Clostridium sporogenes) to produce near-infrared fluorescence from biliverdin and hemin. IMPORTANCE Continued efforts in developing the CRISPR-Cas systems will further enhance our understanding and utilization of Clostridium species. This study demonstrates the development and application of a genome-engineering tool in two Clostridium strains, Clostridium butyricum and Clostridium sporogenes, which have promising potential as probiotics and oncolytic agents. Particular attention was given to the folding of precursor crRNA and the role of this process in off-target DNA cleavage by Cas12a. The results provide the guidelines necessary for efficient genome engineering using this system in clostridia. Our findings not only expand our fundamental understanding of genome-engineering tools in clostridia but also improve this technology to allow use of its full potential in a plethora of biotechnological applications.

The Cas9 protein from Streptococcus pyogenes has been widely utilized for genome engineering in clostridia (7).It recognizes the DNA adjacent to the 5′-NGG-3′ protospacer adjacent motif (PAM) and results in a double-stranded break (DSB) with blunt ends (8).The low efficiency of non-homologous end joining repair in many bacteria and the lethality of unrepaired DNA necessitate the use of homology-directed repair (HDR) (9).Thus, in bacterial CRISPR systems, DNA cleavage selects for the recombination events (mediated by the donor DNA template) that repair the DSB and remove the guide RNA (gRNA) target sequence, akin to selection for resistant colonies with an antibiotic.Various methods have been employed in clostridia to overcome the toxicity of Cas9 to the host, including control of Cas9 expression using inducible promoters or a theophyllinedependent riboswitch (10), as well as selection of alternative versions of Cas9, such as the truncated Cas9 nickase (Cas9n) (7).The large size of the cas9 gene has led some researchers to use a dual-plasmid system, which has been exemplified in solventogenic Clostridium species.One benefit of this approach is the improved cargo capacity of the system, which enables the insertion of larger sequences into the genome (11,12).While genome engineering in clostridia using Cas9 has been applied extensively across multiple species, Cas12a (previously known as Cpf1) is an alternative CRISPR-associated protein that has shown potential advantages over Cas9, including the recognition of T-rich PAMs for clostridia (13) and lower toxicity (14).To date, only in a few solventogenic Clostridium species has the Cas12a protein been introduced for genome engineering (13,(15)(16)(17).
In addition to heterologous Cas proteins, which must be added to the cells as a coding sequence on a plasmid, recent publications have demonstrated the use of endogenous CRISPR-Cas systems for host genome engineering in a few Clostridium species.This approach relies on native expression of an endogenous Cas protein from the host chromosome, removing the need to provide the gene on a plasmid.Higher transformation efficiency is reported using this approach (18)(19)(20)(21)(22). Importantly, prior to applying the endogenous CRISPR-Cas systems, the types of CRISPR array and the requirements of PAM recognition sites in different genotypes of clostridia can vary and need to be characterized.Overall, the development of tools for genetic manipulation in clostridia has advanced significantly in the last 20 years.Further refinement of existing tools, as well as the adoption of new techniques, can support faster development of and greater control over strains for biotechnological applications (23,24).In addition, for efficient genome engineering using CRISPR-Cas systems in bacteria, a set of guidelines for target selection and a better understanding of gRNA quality are necessary (25).
In this study, we hypothesized that the CRISPR-Cas12a system, using two tetracyclineinducible promoters for the expression of Cas12a enzymes (AsCas12a from Acidamino coccus and FnCas12a from Francisella novicida) and crRNAs, would achieve efficient genome engineering in the probiotic C. butyricum (26) and the oncolytic C. sporogenes (27).Our aim was to demonstrate the feasibility of this approach by modifying the genomes of these two Clostridium medical vehicles to produce near-infrared (NIR) fluorescence.Additionally, our study aimed to compare the abilities of the CRISPR-AsCas12a and CRISPR-FnCas12a systems by assessing their efficiency in generating gene (pyrE and spo0A) knockout mutations in C. butyricum and explore guidance for target selection in the CRISPR-Cas12a system.

Tetracycline-inducible systems regulate gene expression in Escherichia coli and C. butyricum
To regulate gene expression in the CRISPR system, we constructed and validated tetracycline-inducible systems in both E. coli and C. butyricum using the glucuronidase (GusA) reporter.A previous tetracycline-inducible GusA expression system (RPF185) was used as the control (28), and two optimized tetracycline-inducible promoters, PIPL12 (29) and Pfet (30), were evaluated.In order to align with the AT-rich genome context of clostridia, the newly codon-optimized Tet repressor (COtetR) was expressed under the control of the miniP4_tU promoter, resulting in the establishment of two tetracy cline-inducible systems named IPL12 and fet, respectively (Fig. S1A).In the absence of the anhydrotetracycline (ATc) inducer, the GusA activity in both E. coli gusA − (Fig. S1B) and C. butyricum (Fig. S1C) was undetectable for all three tetracycline-inducible systems (RPF185, IPL12, and fet).However, in C. butyricum, the RPF185 system exhibi ted undetectable GusA activity even with the addition of 96-ng/mL ATc, while the GusA activity of the IPL12 and fet systems was approximately 24-fold and 31-fold higher, respectively, compared to that of the RPF185 (Fig. S1C).Based on these data, the tetracycline-inducible systems IPL12 and fet were chosen to drive expression of the cas12a gene and the precursor CRISPR RNA (pre-crRNA), respectively, resulting in the construction of the CRISPR-Cas12a system (CRISPR-AsCas12a and CRISPR-FnCas12a) contained in vectors pPIPL12-As/FnCas12a and pPfetAs/Fn-Target_v1 (Fig. 1A).Here, the pPfetAs/Fn-Target_v1 vector was designed to contain a CRISPR array with two direct repeat (DR) sequences and two BsmBI restriction sites, between which a 23-nucleotide target sequence can be ligated (Fig. 1B).Because the Cas12a proteins are known to process the CRISPR array by cleaving upstream of specific hairpin structures formed by the DR (31), the mature crRNA with a single 5′ repeat and the target sequence can be split from the transcript of pre-crRNA, resulting in mature Cas12a/crRNA complex (Fig. 1C).

Optimal folding of pre-crRNA improves mutation efficiency
To evaluate the abilities of the CRISPR-Cas12a system, we initially deleted the pyrE gene (encoding orotate phosphoribosyltransferase) and the spo0A gene (encoding the master regulator for sporulation) in C. butyricum using two different targets that recognize the PAM sequence "TTTA" and using homology arms of approximately 800 bp.Using one target, both AsCas12a and FnCas12a exhibited high mutation efficiency in the deletion of the pyrE (Fig. 2A) or spo0A gene (Fig. 2B).However, another target resulted in no mutation or the presence of colonies containing both wild-type and mutant cells.This occurred in both the CRISPR-AsCas12a and the CRISPR-FnCas12a systems (Fig. 2).This result indicated that mutation efficiency is target sequence dependent for CRISPR-Cas12a systems in C. butyricum.
In order to assess the impact of the target sequence on mutation efficiency, we utilized the RNAfold Web Server (32) to predict the folding of each pre-crRNA in the four aforementioned targets.For the CRISPR-FnCas12a system, we observed the same pre-crRNA folding (complete hairpin structures at each DR) for two target sequences that had high mutation efficiency (Fig. 3A and B).However, another two targets led to the irregular folding of pre-crRNAs, containing different base pairings between the 5′ DR and the target sequence (Fig. 3C and D), which might impair array processing and target recognition by the Cas12a proteins (33).Similar folding patterns of pre-crRNAs were observed in the CRISPR-AsCas12a system (Fig. S2).Additionally, in these same folding of pre-crRNAs, we observed the same value of −9.60 kcal/mol in minimum free energy (MFE), which represents the most thermodynamically stable structure of an RNA molecule under certain conditions (32).We hypothesized that with the MFE value of −9.60 kcal/mol, the possibly optimal folding structure of the pre-crRNA contributed to the high mutant efficiency in the CRISPR-Cas12a system.Thus, each pre-crRNA formed by different targets and predicted with −9.60 kcal/mol in MFE was defined as the Good_Pre-crRNA, while one without such an MFE was defined as the Bad_Pre-crRNA.
Using the CRISPR-FnCas12a system with various targets recognizing different PAMs, all Good_Pre-crRNAs displayed above 90% mutation efficiency in both the pyrE and spo0A gene deletions in C. butyricum.However, the Bad_Pre-crRNAs resulted in variable mutation efficiency in the deletions of pyrE and spo0A in C. butyricum (ranging from 0% to 80%, Fig. 3E and F).No clear correlation was observed between the MFE of the Bad_Pre-crRNAs and the mutation efficiency, suggesting that other factors in the Bad_Pre-crRNA may also affect mutation efficiency.Hence, the folding of the pre-crRNA can affect the mutation efficiency, and the prediction of Good_Pre-crRNA could be beneficial for the use of the CRISPR-Cas12a system in C. butyricum.
To validate this finding across species, the CRISPR-FnCas12a system was utilized to delete the uidA gene (encoding beta-glucuronidase) in E. coli MG1655 RARE (34), using a Good_Pre-crRNA and a Bad_Pre-crRNA recognizing a PAM of ATTA.Similar to the results in C. butyricum, the Good_Pre-crRNA displayed 100% mutation efficiency, while the Bad_Pre-crRNA resulted in most colonies (six out of 14 colonies) containing a mixture of wild-type and mutation cells (Fig. S3).This finding supports the notion that the folding of Good_Pre-crRNA generates high mutant efficiency using the CRISPR-FnCas12a system in a species-independent manner.

CRISPR-FnCas12a is an efficient and flexible gene deletion system in clostri dia
Upon the findings about the Good_Pre-crRNA, to compare the PAM recognition between the CRISPR-AsCas12a and CRISPR-FnCas12a systems, various targets forming the Good_Pre-crRNA in the two CRISPR-Cas12a systems were used to delete the pyrE and spo0A genes in C. butyricum.Using the Good_Pre-crRNA, the CRISPR-AsCas12a system showed high mutation efficiency with PAMs of TTTA, TTTG, and TTTC but low efficiency with TTTT, CTTA, ATTA, and GTTA (Fig. 4A and B), which is in accordance with the known preference of AsCas12a for PAMs of TTTV (V = A/G/C) (35).It has been recently observed that the cleavage efficiency of FnCas12a decreases significantly with a TTTT PAM in several species (36,37).However, our CRISPR-FnCas12a system showed high mutation efficiency when deleting either the pyrE or spo0A genes not only when using PAMs of TTTA, TTTG, TTTC, CTTA, ATTA, or GTTA (Fig. 4A and B) but also when using the TTTT PAM (Fig. 4C).Thus, we concluded that in clostridia, TTN PAM recognition by the CRISPR-FnCas12a system was efficient.The CRISPR-FnCas12a system was chosen for subsequent experiments as it enabled maximum flexibility in the selection of targets that formed the Good_Pre-crRNA in the AT-rich genomes of Clostridium species.

The sacB gene can be used as a negative marker for plasmid curing
To characterize phenotypes following confirmation of the mutation through colony PCR, the CRISPR plasmids involved in the mutation should be cured.We initially observed that the variations of plasmid pPfetFn-Target_v1 were not efficiently curable in C. butyricum (data not shown).To enhance plasmid-curing efficiency in the CRISPR-FnCas12a system, the sacB gene (encoding levansucrase) was cloned into pPfetFn-Target_v1 as a negative marker (38), resulting in plasmid pPfetFn-Target_v2 (Fig. 4D).The addition of 100-g/L sucrose to the cultures containing both the pPIPL12-FnCas12a vector and the derivative vector based on pPfetFn-Target_v2 after overnight incubation did not result in any colonies on agar plates containing the antibiotics thiamphenicol (Tm) or chlorampheni col (Cm), indicating successful curing of the derivative of pPfetFn-Target_v2.On the other hand, colonies containing the pPIPL12-FnCas12a vector could be selected on an agar plate supplemented with the antibiotic erythromycin (Erm) and used for further editing.Similar results were observed in C. butyricum, C. sporogenes, and E. coli (Fig. S4).Thus, the inclusion of the sacB-negative marker was found to be effective in promoting plasmid curing in the CRISPR-FnCas12a system.

CRISPR-FnCas12a-mediated gene deletions result in corresponding pheno type changes
In order to evaluate whether CRISPR-mediated gene deletions resulted in the expected outcome, we characterized the corresponding phenotypes of the mutations.For the characterization of the pyrE gene deletion, we evaluated cell growth in the presence of 5-fluoroorotic acid (FOA).This approach is based on the observation that FOA is toxic to clostridia harboring a functional pyrE pathway (5).As expected, C. butyricum harboring the deletion of pyrE (CB_ΔpyrE) was able to grow on agar plates supplemented with 1-mg/mL 5-FOA, whereas C. butyricum wild type (CB_WT) was unable to grow due to the presence of the pyrE pathway (Fig. S5A).For the characterization of the spo0A gene deletion, we performed a sporulation assay.Using this assay, we demonstrated that C. butyricum harboring the spo0A deletion (CB_Δspo0A) was unable to form spores, whereas the CB_WT strain was able to generate heat-resistant colonies of about 5 × 10 5 CFU/mL (Fig. S5B).Additionally, in the GusA assay, E. coli MG1655 RARE harboring the deletion of uidA (RARE_ΔuidA) was unable to exhibit detectable GusA activity, while the wild-type strain RARE_WT could (Fig. S5C).These results demonstrate that gene deletions using the CRISPR-FnCas12a system lead to corresponding phenotype changes.

Gene integration efficiency is affected by the size of homology arms in donor DNA templates
The efficiency of recombination of the donor DNA template is a crucial factor in HDR-mediated CRISPR systems (39), and the appropriate size of homology arms may vary depending on the species or systems.In the experiments of gene deletion carried out in E. coli and C. butyricum as described above, a 0.8-kb homology arm was selected.Using the CRISPR-FnCas12a system, to further examine the effect of homology arm size on recombination efficiency in C. butyricum, we truncated the size of the homology arm in the donor DNA template from 1.5 to 0.25 kb (Fig. 5A).Using a 0.25-kb size of homology arm to integrate a 2-kb size of gusA expression cassette into C. butyricum did not result in the desired mutation.The mutation was observed when the homology arm size was increased from 0.5 to 1.5 kb.The mutation efficiency reached 100% with 1.0-and 1.5-kb homology arms.Similar results were obtained using different targets (Fig. 5B).These data indicate that the efficiency of gene integration using the CRISPR-FnCas12a system is dependent on the homology arm size in the donor DNA template and that a size of at least 0.5 kb is necessary for the recombination to acquire the desired mutation in C. butyricum.Of note, using the GusA assay, we characterized the mutation phenotype of C. butyricum harboring the integrated gusA expression cassette (CB_INgusA), which showed an increase in GusA activity compared to CB_WT (Fig. S5C).

C. butyricum and C. sporogenes can be engineered to produce NIR fluores cence
As depicted in Fig. 6, the construction of plasmids following the guidelines for tar get selection and donor DNA template design can be accomplished within about 4 days.Subsequently, genome-engineered clostridia can be generated using the CRISPR-FnCas12a system within about 1 week, and then the phenotype of the plasmid-cured mutations can be characterized.
To demonstrate the versatility and efficiency of the tetracycline-inducible CRISPR-FnCas12a system across different Clostridium species, we set out to engineer C. butyricum and C. sporogenes to produce NIR fluorescence, which might offer a deep penetration in the anaerobic tracking of clostridia (40).The IFP2.0 gene, derived from Rhodopseudomo nas palustris, was used as it codes for a NIR fluorescent protein that requires biliverdin (Bv) as a chromophore cofactor (41).The IFP2.0 gene expression cassette was codon  S4).The desired changes to the genome are confirmed by Sanger sequencing; furthermore, the phenotype can be functionally characterized.
optimized and efficiently integrated into the genomes of C. butyricum (CB001 strain) and C. sporogenes (CS001 strain), as shown in Fig. 7A.
The green bile pigment, Bv, is catabolized from hemin by the hemO gene (encoding hemin oxygenase).The hemO gene from C. novyi, driven by a strong promoter Pthl79% −26_fU, was codon optimized and efficiently integrated into the genomes of the CB001 and CS001 strains, resulting in the CB003 and CS003 strains, respectively (Fig. 7B).The wild-type CB_WT and CS_WT strains did not exhibit detectable NIR fluorescence, with or without the addition of either Bv or hemin (Fig. 7C and D).The CB001 and CS001 strains showed NIR fluorescence only with the addition of 10-µM Bv, and the highest fluorescence intensity was observed at around the 6-hour time point (Fig. 7E and F).Due to the toxicity of high concentrations of hemin in bacteria (42), we determined the appropriate concentration of hemin in the CB003 and CS003 strains (Fig. S6).With the addition of 7.5-µM hemin, the CB003 strain displayed NIR fluorescence along with cell growth (Fig. 7G), while the CB_WT and CB001 could not exhibit obvious growth until the 24-hour time point without NIR fluorescence (Fig. 7C and E).These data suggest that the integration of hemin oxygenase could improve the tolerance of hemin toxicity in C. butyricum.Furthermore, with the addition of 2.5-µM hemin, the CS003 strain displayed NIR fluorescence along with normal cell growth (Fig. 7H).When supplied with either hemin or Bv, at the 6-hour time point, confocal imaging revealed NIR fluorescent cells in the CB003 and CS003 strains (Fig. 8), while only green auto-fluorescent cells were observed in the wild-type strains (Fig. S7).These data demonstrate that the genomes of C. butyricum and C. sporogenes could be modified by the tetracycline-inducible CRISPR-FnCas12a to produce NIR fluorescence from hemin and Bv.

DISCUSSION
Clostridium offers a broad range of potential applications in biotechnology, whether for medical or industrial applications.However, genetic manipulation in clostridia is still challenging due to species-specific nuances (43), and the CRISPR-Cas tools in clostridia are not as refined as in model organisms (7).In this study, we achieved successful implementation of the CRISPR-Cas12a system in two Clostridium sensu stricto species, C. butyricum, and C. sporogenes, utilizing tetracycline-inducible systems.Currently, many Clostridium studies have designed multiple single-guide RNAs (sgRNAs) and gRNAs in the CRISPR-Cas systems to acquire mutations efficiently, which is limited for the rapid genome engineering in Clostridium research (7).Unlike the sgRNA selection in CRISPR-Cas9 systems by various computational tools (25), the identification of gRNAs with high mutation efficiency remains a challenge for CRISPR-Cas12a systems in diverse species (31,44,45).Initially, we used the web tool CRISPOR (46) to select target sites for pre-crRNA, with TTTV as the PAM for both AsCas12a and FnCas12a.Despite this effort, some targets still exhibited low efficiency in mutation generation.Inspired by the pre-crRNA processing of Cas12a, we determined that predicting the specific folding of pre-crRNA by utilizing the RNAfold Web Server (32) could enhance the mutation efficiency in C. butyricum.By deleting the uidA gene in the model microbe E. coli, we suggest that selecting targets based on pre-crRNA folding predictions may improve the efficiency of CRISPR-Cas12a systems across various species.There is a recent observation in Clostridium beijerinckii that some target sequences in the CRISPR array result in the low efficiency of their CRISPR-FnCas12a system (17).
In previous studies that focused on the impact of PAM selection on Cas12a-mediated genome engineering, the design of the CRISPR array was different and pre-crRNA folding was not analyzed in detail.This could explain inconsistent reports on the performance of FnCas12a when "TTT" is selected as the PAM sequence.The widespread availability of "TTN" as a PAM sequence in Clostridium genomes increases the number of loci that can be targeted but may also raise concerns over off-target effects (47).Predicting the folding of pre-crRNA should be a necessary aspect to be considered in Clostridium CRISPR-Cas12a systems, to carefully select targets and reduce potential off-target effects.Furthermore, high-GC targets have been found to impair the activity of the Cas12a array (31).Given the low GC content in clostridia genomes, we used targets with an average GC content of below 50% (Table 1), which may not be a critical aspect of exploring CRISPR-Cas12a systems in clostridia.However, we encountered difficulty determining why some targets forming the "bad" folding of pre-crRNA were still efficient in our study.We hypothesize that the frequency of "bad" folding in pre-crRNAs could differ due to the varying stability of base pairing (48), resulting in on-target effects of Bad_Pre-crRNAs.Therefore, the accurate prediction of RNA folding would be beneficial for investigating off-target effects, and we encourage RNA folding to be considered in computational tools to predict efficient gRNAs (25).Recently, several dCas12a-based CRISPR interference systems have been developed for tuning gene expression in clostridia (13,47,49).In our later work, we would like to investigate the impact of the folding of pre-crRNAs on the efficiency of gene repression.
Prior to engineering the genome of clostridia for NIR fluorescence production, we evaluated three oxygen-independent fluorescent proteins: CreiLOV (50), UnaG (51), and IFP2.0 (41), which use natural substrates as cofactors (flavin, bilirubin, and Bv, respec tively).However, during confocal imaging in the 488-nm channel, we observed high levels of green auto-fluorescence in C. butyricum, which made it difficult to distinguish between C. butyricum expressing CreiLOV or UnaG with and without the addition of flavin or bilirubin.In contrast, C. butyricum expressing IFP2.0 exhibited low background auto-fluorescence in the 635-nm channel, making it easier to distinguish the addition of Bv (Fig. S7).The green auto-fluorescence has been observed in Clostridioides difficile, the mechanism of which is still unclear (52).In addition, various Bv-based red fluorescent proteins have been developed continuously (53), which would serve as alternatives to IFP2.0.After integrating IFP2.0, we initially integrated the hemO gene driven by the promoter Pthl_fU into the CB001 and CS001 strains, obtaining the CB002 and CS002 strains.We observed that hemin led to growth inhibition in the CB002 and CS002 strains (Fig. S6), and, thus, we improved the expression of the hemO gene by substituting the promoter Pthl_fU with a stronger promoter, Pthl79%-26_fU (38,54).The toxicity of hemin in bacteria is multifactorial, and our understanding of it is still incomplete (42,55).In some Clostridium species, hemin oxygenase is involved in enhanced aero-toler ance by degrading hemin to Bv (56), which could improve the colonization capacity in Clostridium-mediated therapy (3).In order to avoid the external supply of hemin, we attempted to integrate the hemin biosynthetic pathway into the genome of C. sporogenes using the CRISPR-FnCas12a system (Fig. S8A).Inspired by the metabolic engineering of E. coli for hemin production (57,58), we introduced three polycistronic expression cassettes into the genome of the CS003 strain with high mutation efficiency: hemLA (2,766 bp), hemNG (2,212 bp), and hemEH (2,320 bp) resulting in the creation of strain CS006 (Fig. S8B).Despite the addition of the required Fe 2+ ion and the electron acceptor menaquinone of HemG ( 59), the final strain CS006 did not exhibit detectable NIR fluorescence along with cell growth (Fig. S8C and D).This may be due to a lack of metabolic flux into the hemin biosynthetic pathway.Overexpression of critical genes to increase the metabolites available for this pathway could provide a solution.This could be achieved by increasing the copy number of critical genes in the genome or on a high copy number plasmid, as previously reported (60).However, to date, there are no reports of de novo hemin biosynthesis in clostridia.The lack knowledge on the biology of non-model microbes like clostridia, especially when compared to E. coli, presents a major challenge for metabolic engineering and synthetic biology (24).Overall, this study demonstrates that the CRISPR-FnCas12a system can be a powerful gene integration tool in clostridia.This work introduces the concept of endogenous fluorescence in recombi nant clostridia, which will enable visualization and tracking of the bacteria.The substrate for the fluorescence reaction (hemin) occurs naturally and can be obtained from dietary sources and from hemoglobin and Bv, a pigment that produced in the intestine.This is an obvious advantage for the application of genetically engineered clostridia in the medical setting, such as intestinal probiotics and intratumoral living therapeutics (3,61,62).
In conclusion, this study introduces a tetracycline-inducible CRISPR-Cas12a system that enables robust genome engineering in two Clostridium medical vehicles.We achieved gene deletions and heterologous gene integrations with a high efficiency ranging from 85% to 100% using the CRISPR-FnCas12a system (Table 1), which can be one of the most powerful CRISPR-Cas systems in clostridia.The study highlights the significance of RNA folding prediction in analyzing the off-target effects of Cas12 proteins in various species.Further studies on the NIR fluorescent clostridia provide an opportunity for anaerobic tracking in Clostridium-mediated living therapeutics and emphasize the importance of understanding this non-model microbe for biotechnologi cal applications using genome-engineering tools.

Plasmids construction
The plasmid details are listed in Table S1, and the details of the primers used are in Table S2.For comparison of tetracycline-inducible systems using GusA assays, two gBlock fragments (COtetR-PIPL12 and COtetR-Pfet) were synthesized by Integrated DNA

Target selection
To select targets for the Cas12a proteins, we identified regions in the target strand that had Cas12a PAMs (TTTV or TTN).We used CRISPOR (46) and NCBI-BLAST to ensure that the 23-nucleotide target sequence downstream of the 5′ PAM motif was specific to the region.Then, we used RNAfold Web Server (32) to predict the pre-crRNA folding and MFE value.

Calculation of mutation efficiency
To calculate mutation efficiency, we transformed the plasmids pPIPL12-AsCas12a and pPIPL12-FnCas12a into clostridia and confirmed trans-conjugants by Sanger sequencing (GENEWIZ).We next transformed the vectors containing the target and the donor DNA template into the clostridia containing either pPIPL12-AsCas12a or pPIPL12-FnCas12a.Then, at least three trans-conjugants were re-streaked on PYTG agar plates containing D-cycloserine, Tm, Erm, and ATc.Finally, we randomly picked 14 colonies and performed colony PCR.The mutation efficiency was calculated by dividing the number of mutations detected by colony PCR by the total number of colonies in the screen, multiplied by 100.The PCR products of mutations were verified by Sanger sequencing (GENEWIZ).

Detection of fluorescent intensity
Overnight cultures of Clostridium strains were sub-cultured into fresh PYTG media (2:100) with substrates added.For each time point, 1 mL of the culture was washed once with PBS by centrifugation and suspended in 1 mL of PBS.The fluorescent intensity of 100 µL of the suspension was measured on a 96-well polystyrene plate (P8741, Sigma) using a SpectraMax iD3 Multi-Mode Microplate Reader with excitation and emission wavelengths of 660 and 730 nm, respectively.The rest of the suspension was used to measure the OD 600 .

Confocal imaging
To prepare samples for confocal imaging, Clostridium strains were sub-cultured into fresh PYTG media at a 1:100 dilution and grown with substrates for 6 hours.After washing the cells once in PBS, they were fixed in 4% formaldehyde (28,906,Thermo Scientific) in the dark for 15 minutes, followed by two additional washes in PBS.The fixed cells were mixed with an equal volume of fluorescent mounting medium (S3023, Dako) and mounted on a glass slide with a cover slip for overnight incubation.The confocal images of the cells were captured using a Leica SPE confocal laser scanning microscope (Leica Microsystems GmbH) equipped with diode lasers of 488 and 635 nm and oil immersion objectives (×63, numerical aperture = 1.4) (67).Three scans were performed for each image, and the images were analyzed and exported using Leica Application Suite X (LAS X).

FIG 2
FIG 2 Deleting the pyrE and spo0A genes in C. butyricum using the CRISPR-Cas12a system.Colony PCR to check the deletions of genes pyrE (A) and spo0A (B) in C. butyricum by the CRISPR-AsCas12a (green) and CRISPR-FnCas12a (blue) systems.The recognized PAMs of the target sequences were highlighted in red.The PCR products of the pyrE mutation and the wild type were 2,172 and 2,860 bp, respectively.The PCR products of the spo0A mutation and the wild type were 1,901 and 2,751 bp, respectively.The Quick-Load Purple 1-kb Plus DNA (N0550L, NEB) was used as the molecular weight standard.1-14, PCR products from single colonies; W, PCR products from the wild-type genome.

FIG 4
FIG 4 Efficient and flexible gene deletion in clostridia by the CRISPR-FnCas12a system and the inclusion of the SacB-negative marker for efficient plasmid curing.Using various Good_Pre-crRNAs recognizing different PAMs, the mutation efficiency of deleting pyrE (A) and spo0A (B) genes in C. butyricum by the CRISPR-AsCas12a (green) and CRISPR-FnCas12a (blue) systems was determined.(C) Using targets recognizing TTTT PAM and colony PCR to check deletions of pyrE and spo0A genes in C. butyricum by the CRISPR-FnCas12a system.(D) The schema of the general vector pPfetFn-Target_v2.The inclusion of the SacB negative marker was highlighted in yellow.

FIG 5
FIG 5 Donor DNA templates affect gene integration efficiency.(A) The schema of donor DNA templates with different sizes of homology arms to integrate the GusA expression cassette (about 2-kb size) into C. butyricum.(B) In the CRISPR-FnCas12a system, use colony PCR to check the integration of the GusA expression cassette in C. butyricum using two different targets.The sizes of the PCR products of the mutation harboring the integration of the GusA expression cassette and the wild type were 5,426 and 3,536 bp, respectively.The GeneRuler 1-kb DNA ladder (SM0311, Thermo Scientific) was used as the molecular weight standard.1-14, PCR products from single colonies; W, PCR products from the wild-type genome.

FIG 6
FIG 6 Procedure for genome engineering in clostridia by the CRISPR-FnCas12a system.The targets and donor DNA templates are ligated into pTarget (the general vector pPfetFn-Target_v2) and confirmed by Sanger sequencing (4 days).The engineered clostridia are generated as follows: the confirmed vector pTarget is transformed into the clostridia containing pCas (the pPIPL12-FnCas12a vector) (Days 1 and 2); the trans-conjugants are re-streaked on new agar plates containing the ATc inducer (Days 3 and 4); the correct mutations are confirmed by colony PCR and are cultures overnight with the addition of sucrose for plasmids curing (Day 5); an overnight culture of the correct mutations is serially diluted and plated (Day 6); the presence of each plasmid in mutants is determined by their ability to grow on antibiotic selection plates (Day 7).Clones that can grow on Erm plates are used for subsequent rounds of genetic engineering (Fig.S4).The desired changes to the genome are confirmed by Sanger sequencing; furthermore, the phenotype can be functionally characterized.

FIG 7
FIG 7 Genome engineering in C. butyricum and C. sporogenes to produce NIR fluorescence from Bv and hemin.(A) Colony PCR to check the integration of the IFP2.0 expression cassette in wild-type strains of C. butyricum and C. sporogenes, obtaining CB001 (size of mutation/wild type, 4,580/3,536 bp) and CS001 (size of mutation/wild type, 3,506/2,475 bp) strains.(B) Colony PCR to check the integration of the HemO expression cassette with promoter Pthl79%-26_fU in CB001 and CS001 strains, obtaining CB003 (size of mutation/wild type, 3,133/2,338 bp) and CS003 (size of mutation/wild type, 3,119/2,343 bp) strains.With or without the addition of Bv or hemin, the growth curve and the intensity of NIR fluorescence (A.U. in 660/730 nm of Ex/Em) in wild type CB_WT (C) and CS_WT (D) strains and the CB001 (E), CS001 (F), CB003 (G), and CS003 (H) strains.Bv, 10 µM in all strains; Hemin, 7.5 µM in CB_WT, CB001, and CB003 strains; and 2.5 µM in CS_WT, CS001, and CS003 strains.The data represent the mean ± SD of three biological replicates.

FIG 8
FIG 8 Confocal imaging in 488 635 nm and merged channels of the CB003 and CS003 strains with the addition of Bv or hemin.(A) The CB003 with an addition of 10-µM Bv; (B) the CB003 with an addition of 7.5-µM hemin; (C) the CS003 with an addition of 10-µM Bv; and (D) the CB003 with an addition of 2.5-µM hemin.Scale bar, 25 µm.

TABLE 1
Targets used in the CRISPR-FnCas12a system